Production of amino acids and enzymes used therefor

ABSTRACT

A process for producing α-amino acid from starting material comprising α-amino amide enantiomers (A) and (B) is such that enantiomer (A) is converted preferentially over enantiomer (B) so that a time independent excess of at least 90%, preferably at least 98% is given. The reaction is catalyzed by an amidase which may in particular be produced by specific Rhodococcus species.

This application is a 371 of PCT/GB97/02031 Jul. 25, 1997.

The invention relates to new processes of enantioselective conversion ofα-amino amides, α-methyl amides and α-aminomethyl amides to theircorresponding acids and to new microorganisms and enzymes useful inthese processes.

It is known that α-amino amides may be converted to α-amino acids byhydrolysis. This hydrolysis may be catalyzed by an amidase enzyme. It isalso known to convert α-amino nitriles to α-amino acids by firstconverting the nitrile to an amide by its hydration. Thisnitrile-to-amide hydration may be carried out using a nitrile hydrataseenzyme catalyst.

It is known that these transformations may be carried is out in anenantioselective fashion (that is, the α-amino acid produced has anexcess of one enantiomer) by choice of suitable enzymes. For instance,Bauer et al in Appl. Microbiol. Biotechnol. (1994) 42:1-7 describes theconversion of α-amino phenyl acetonitrile to α-amino phenyl acetic acid.The conversion is catalyzed by a strain of Agrobacterium tumefaciens.The nitrile is converted in almost stoichiometric amounts to the amide.The amide is then slowly hydrolyzed to the acid. The S enantiomer ispreferentially formed. The best enantiomeric excess (97%) appears to beachieved after 43% conversion of the amide.

It appears that it is necessary to terminate the reaction at this pointin order to achieve the high enantiomeric excess, ie the conversion doesnot give time-independent enantioselectivity.

This requirement to stop the reaction at a particular point in order toachieve the best enantiomeric excess is well known in enantioselectivehydrolysis of amides to acids. In general, known catalysts of the abovementioned amides to their corresponding acids hydrolyse one amideenantiomer more rapidly than the other and eventually tend to convert asignificant amount of the less preferred enantiomer.

Other conversion reactions have been described which appear to give highconversion and enantioselectivity, but do not appear to exhibit tameindependent enantioselectivity. For instance, EP-A-332,379 disclosesproduction of various amino acids. These are produced from a nitrile byexposure to various microorganisms. It is not stated whether theeffective enzyme catalyst is a nitrilase or a combination of nitrilehydratase and amidase. It is stated that it is essential to carry outthe reaction either at a pH between 8 and 12 or in the presence of analdehyde. U.S. Pat. No. 4,080,259 describes amidase enzymes which effecthydrolysis of various amides to produce amino acids. Where the pH of thereaction mixture is given it appears to be from around 8 to around 10.U.S. Pat. No. 4,366,250 and FR-A-2,626,287 describe enzymic hydrolysisof various natural amino acids. U.S. Pat. No. 3,971,700 describesselective hydrolysis of phenyl glycine amide to give phenyl glycine.None of these references alleges that the reaction described gives timeindependent enantioselectivity.

U.S. Pat. No. 5,248,608 describes an enantioselective hydrolysis ofvarious α-substituted carboxylic acid amides. The hydrolysis is carriedout using an amidase enzyme produced by Ochrobactrum anthropi or aKlebsiella sp. The citation alleges that very high conversion andenantiomeric excess are obtained. It explains that the general theoryregarding enantioselective conversions described in past publicationsapplies to the process described in U.S. Pat. No. 5,248,608. Thepublications referred to are those which rely upon stopping the reactionat a particular time in order to obtain a high enantiomeric excess i.e.reactions which do not give time independent enantioselectivity.

This document describes various examples. Some examples are ofhydrolysis of α-amino amides but the majority are of other α-substitutedamides. Only one of the examples demonstrates time independentenantioselectivity. This single example demonstrates hydrolysis of anN-hydroxy substituted α-amino amide.

Some examples demonstrate hydrolysis of α-amino amides, but none ofthese show time independent enantioselectivity. Further, the maximumtime for reaction which is demonstrated in these examples is 8 hours.The reactions are all carried out as batch reactions.

U.S. Pat. No. 5,215,897 also describes an enantioselective hydrolysis ofamino acid amide, which produces mainly L-amino acid. Again the onlyreactions described are batch reactions which are carried out for amaximum of three hours. The yield in the majority of the reactions isbelow 50% of the starting mixture and there is no demonstration that thereaction is or could be such that it gives time independentenantioselectivity.

It would be desirable to be able to produce α-amino is amides having ahigh enantiomeric excess. This is particularly desirable for theproduction of enantiomerically pure unnatural α-amino acids. It wouldalso be desirable to achieve such enantiomeric purity together with highconversion of the relevant amide enantiomer. It would also be desirableto be able to do this in a manner which provides production processeswhich are conveniently adaptable to an industrial scale.

According to a first aspect of the invention we provide a process ofconverting an α-amino amide to an α-amino acid, comprising conducting aconversion reaction catalyzed by an amidase enzyme, in which the α-aminoamide starting material comprises amide enantiomers (A) and (B) and inthe conversion reaction enantiomer (A) is converted preferentially overenantiomer (B),

Characterized in that the amidase enzyme is capable of convertingenantiomer (A) such that it gives an enantiomeric excess of at least 90%independently of the conversion time.

In this specification, when we say that the enantiomeric excess is givenindependently of conversion time, we mean chat the high enantiomericexcess is retained throughout the time there is sufficient of enantiomer(A) to dominate the reaction, that is until most of enantiomer (A) isconverted. For instance this can be up to 90% conversion of enantiomer(A). Generally time independent enantiomeric excess is maintained up to95% and often 100% conversion of enantiomer IA). In some cases timeindependent enantiomeric excess is remained beyond 100% conversion ofenantiomer (A), but this is not essential. The enantiomeric excess isthat of the acid product.

Thus in the invention we achieve high selectivity for one enantiomer. Inthe majority of known processes the enantiomeric excess in the productvaries as the conversion reaction progresses and it is often necessaryto stop the reaction at a suitable point in order to obtain the bestenantiomeric excess. In the invention, however, we achieve highenantiomeric excess at all times during the reaction, ie independentlyof the conversion time. This is believed to be due to very highselectivity of the amidase for a single enantiomer of the startingmaterial. In known reactions it is usually observed that the amidaseconverts one enantiomer faster than the other, so that as the reactionprogresses the enantiomeric excess tends to decrease. The presentprocess is selective to such an extent that the enantiomeric excessremains at least 90% throughout the reaction.

The starting material in the conversion reaction of the process of theinvention is an α-amino amide. The amide is chosen so that it gives onhydrolysis the required α-amino acid.

Suitable α-amino amides used as starting materials have the formula I asfollows:

In this preferred formula R is suitably alkyl, cycloalkyl, alkenyl,cycloalkenyl, aryl, alkaryl, aralkyl, R¹NHCOR¹, R¹CONHR¹, SO₂R¹ orSO₂NHR¹ in which R¹ is alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl,alkaryl or aralkyl, or substituted versions of any of these. Inparticular, R can be C₄ to C₉, for instance C₄ to C₇, linear or branchedalkyl or alkenyl, cyclic alkyl or alkenyl, phenyl, or substituted phenylin which the substituent is selected from para-CH₃, meta-CH₃, ortho-CH₃,para-CF₃, para-Et, para-(CH₃)₃C, para-Cl, para-CH ₃(CH₂)₃O and para-OH.These substituents, and others, in the meta and ortho positions can beused.

According to the formula I it is particularly preferred that if R isalkyl or alkenyl it, is C₄ to C₉, for instance C₄ to C₇, linear alkyl oralkenyl or, in particular, cyclic alkyl. If R is phenyl it is preferredthat it is a substituted phenyl, in particular one in which thesubstituent is selected frown para-CH ₃, meta-CH₃, para-CF₃, ortho-CH₃,para-Et, para-(CH₃)₃C, para-Cl and para-CH₃(CH )₃O.

The conversion reaction thus produces an α-amino acid of the formula II,as follows:

The process of the invention is particularly suitable for the productionof unnatural amino acids.

The amide starting material comprises enantiomer (A) and enantiomer (B)Enantiomer (A) is that which it is desired to convert preferentiallyinto the corresponding acid during the conversion reaction. Enantiomer Ais usually the L amino acid amide, which gives the L amino acid onconversion.

Enantiomers (A) and (B) can be present in substantially equimolaramounts, that is the starting material is preferably a racemic mixture.However, the process of the invention can also be applied to startingmaterials which have an enantiomeric excess of greater than zero. Theprocess may be applied to starting materials in which enantiomer (A) isin excess and to those in which enantiomer (B) is in excess.

The process is particularly useful in systems in which the enantiomer(B), which is not desired to be converted preferentially into thecorresponding acid, is present in excess. In some types of reaction theratio of enantiomer (B) to enantiomer (A) will steadily increase duringthe reaction as the amount of enantiomer (A) is reduced by conversion tothe corresponding amino acid. In other types of reaction the supply ofenantiomer (A) may be periodically or continuously replenished so thatthe ratio of enantiomer (A) to enantiomer (B) varies over the course ofthe reaction both upwards and downwards. Preferably the amount ofenantiomer (B) is at least 125% or 150%, preferably at least 200%, morepreferably at least 250%, of the amount of enantiomer (A) for at leastsome of the duration of the reaction, preferably at least 30 minutes,more preferably at least 1 or 2 hours.

The amide starting material may be produced in any suitable manner. Itis preferred that the amide is produced by an enzyme catalyzed reaction,especially from nitrile starting material. Therefore in preferredprocesses of the invention an initial step comprises the conversion ofan α-amino nitrile to its corresponding α-amino amide, catalyzed by anitrile hydratase enzyme. The nitrile hydratase enzyme may be one whichpreferentially converts one nitrile enantiomer over the other. Generallyhowever the nitrile hydratase acts non-selectively and converts bothnitrile enantiomers at substantially the same rate.

The amidase enzyme used in the process of the invention is one which iscapable of preferentially converting enantiomer (A) so that it gives anenantiomeric excess of at least 90% independently of the conversiontime.

Preferably the time-independent enantiomeric excess is at least 95%morepreferably at least 97%, and in particular at least 98%.

The conversion reaction is preferably carried out to such an extent thatat least 70% preferably at least 80 or 90%, more preferably at least 95or 98% and even substantially 100% of enantiomer (A) has been convertedto its corresponding acid.

In the conversion reaction of the process of the invention the amidaseis chosen so that it converts only very small amounts of enantiomer (B).Preferably at the end of the conversion reaction not more than 10% morepreferably not more than 5% or even 2% and often substantially none ofenantiomer (B) has been converted to its corresponding acid. Inparticular, in the process of the invention it is preferred that theamidase is such that it results in less than 10%, preferably less than5% or even 2%, more preferably substantially no conversion of enantiomer(B) even when the concentration of enantiomer (A) has fallen belowdetectable levels.

Any conditions suitable for carrying out the amide-to-acid conversionreaction may be used. Conditions are generally chosen to be optimal forthe action of the amidase enzyme. The enzyme used in the invention isone which is capable of carrying out the specified conversion. In somecircumstances it may be desirable to operate the conversion reactionunder conditions which do not give optimal enantiomeric excess. Forinstance it may be desirable in optimising the economics of theconversion to operate at an elevated temperature. Some enzymes tend toexhibit diminished selectivity to their substrate as temperatureincreases. Therefore in some cases it may be necessary to strike abalance between selectivity and productivity. However, it is essentialin the invention that the amidase enzyme is capable of carrying out thespecified conversion under at least some conditions. Preferably theenzyme is capable of carrying out the highly selective conversion at atemperature of from 10 to 50°, often around 30° C. An advantage of theextremely selective enzymes used in the invention is chat they can beoperated at higher temperatures than standard amidase enzymes, thusincreasing productivity, whilst retaining higher selectivity thanstandard amidase enzymes.

Temperature in the conversion reaction is usually from 10 to 50° C.,preferably 15 to 35° C., often 20 or 30° C.

Any suitable pH may be used for the conversion reaction. Preferablyhowever a pH below 8 is used, in particular 6 to 7.5 or 8 and oftenaround 7. This is in contrast with the process of EP-A-332,379, in whichit is essential to use pH of 8 to 12 if an aldehyde is not present.According to the process of the present invention it is possible tocarry out the conversion reaction at a pH below 8 in the absence of analdehyde.

Starting material may be included in the reaction mixture in any desiredconcentration. Concentrations of 1 mM to 2M are particularly suitable,for instance 5 mM to 1M, often around 10 to 50 mM, in particular up to20 mM.

The amidase enzyme may be included in the reaction mixture in anysuitable form. The amidase enzyme is generally produced by amicroorganism. The amidase enzyme may be used in for instance the pureform, having been extracted from a cultured microorganism before use asa catalyst. The extraction method used should ensure that the activityand stability of the amidase are not lost. It may also be used in asemi-pure form, for instance as liquid culture or as a bacterial cellfraction such as intact cells or crushed cells. It may be used in theform of crude, impure enzyme solution. IL may be supported orimmobilised on a carrier, such as a cross-linked polymeric matrix, egcross-linked polyvinyl alcohol or cross-linked polyacrylamide. It may beused in the form of non-swollen particles having surface-bound enzyme.Preferably it is used in the form of intact bacterial cells, free orsupported in a cross-linked polymeric matrix.

The duration of the conversion reaction can vary according to thereagents used, conditions chosen and type of reaction. The reaction maybe a batch reaction. In a batch reaction all reagents are combined atthe beginning of the conversion reaction and allowed co react. In thiscase it is convenient that the reaction continues for up to for instancefour hours, for instance 1 to 3 hours.

Alternatively the reaction may be carried out as a fed batch orcontinuous reaction. It is one of the advantages of the reaction thatdue to the time independent enantiomeric excess which is obtained suchtypes of reaction are feasible. These are particularly preferred for useon an industrial scare.

In a fed batch reaction, reactants are placed in a reaction vessel atthe beginning of the reaction and allowed to react During the reactiontime concentration of starting material will decrease as it is convertedto the final product. Therefore during the reaction additional startingmaterial is added. Normally a lower and an upper limit on theconcentration of starting material are both set and the startingmaterial is initially supplied so as to have a concentration at theupper level and allowed to fall to the lower level. Additional startingmaterial is then supplied so as to raise its level to the upper limitand so on until the reaction has proceeded to an extent that sufficientproduct is produced.

In a continuous reaction the reagents are kept in steady state withsupply of starting material at the same rate as removal of the reactionmixture from the reaction vessel. In such systems it is particularlydesirable to be able to provide a reaction mixture which contains a verylow level of starting material and a very high level of reactionproduct, so that the removed reaction mixture does not need to undergoextensive purification procedures. This is again possible with theprocess of the invention.

In both fed batch and continuous processes it is a particular advantageof the invention that it may be carried out in the presence of highconcentrations of starting material. Preferably during the reaction theratio of product amino acid to enantiomer A starting material becomes atleast 5:4, preferably at least 3:2 and more preferably at least 2:1 or3:1. Preferably this ratio is maintained in the reaction for at least 30minutes, more preferably at least 1 or 2 hours.

Fed batch and continuous reactions are also particularly suited to beingcarried out over extended periods of time, which can be highly desirableon a commercial scale. For instance the conversion reaction may continuefor at least eight hours, preferably at least nine hours, and maycontinue for at least 12 to 15 hours and even up to 24 or 48 hours ormore.

It is particularly desirable in the invention that the only enzymecatalyst present in the reaction mixture is an amidase (optionallytogether with a nitrile hydratase, if this is used to produce the amidestarting material). In particular it is preferred that no racemase ispresent in the conversion reaction mixture. Some conversions are knownin which racemase must be used in combination with an amidase which issaid to be enantioselective. However these must be carried out in thepresence of a racemase enzyme, which we believe continuously convertsthe enantiomer which is not preferentially converted into a racemicmixture. This is because, we believe, the conversion of amide to acid isnot time independent and as the concentration of the unpreferredunreacted enantiomer builds up, conversion of that begins to thecorresponding amino acid.

Whatever reaction type is used it is preferred that at the end of thereaction at least 80%, preferably at lest 90 or 95%, more preferably atleast 98% and often substantially 100%, of enantiomer A has beenconverted to its corresponding acid.

Measurement of the enantiomeric excess achieved at the end of theprocess of the invention can be carried out in any standard manner.

The reaction conditions given for the amide-to-acid conversion reactionare also generally applicable to a nitrile-to-amide conversion reactionif this is included in the process of the invention and when catalyzedusing a nitrile hydratase enzyme, with the exception that concentrationsof 1 to 100 mM, particularly 5 to 50 or 20 mM, of nitrile are preferred.

At the termination of the process of the invention the enantiomericallypure amino acid will have been produced from enantiomer (A). Largeamounts of unconverted enantiomer (B) may also remain in the reactionmixture. In is some cases the enantiomerically pure (B) amide may berequired for use and is also a product of the conversion reaction. Theconverted enantiomer (A) and unconverted enantiomer (B) are generallyseparated. The enantiomerically pure amino acid derived from enantiomer(A) can then be used as desired. Unconverted enantiomer (B) may also beused as required. If it is desired to obtain the enantiomerically pureamino acid derived from enantiomer (B) then chemical hydrolysis ofunconverted enantiomer (B) can be carried out in known manner. Ifenantiomer (B) is not required, it is particularly efficient to subjectit to racemisation and to include the resultant racemic mixture ofenantiomers (A) and (B) in a further batch of amide starting materialfor the process of the invention. Racemisation can be carried outchemically. Alternatively it can be carried out with the use of aracemase enzyme. If enantiomer (B) is subjected to racemisation and theresultant racemic mixture of enantiomers (A) and (B) is then furtherreacted, conversion of up to loot; of the original starting material canbe achieved.

In the process of the first aspect of the invention the amidase enzymeis preferably produced by the microorganism of genus Rhodococcus whichhas been deposited at the National Collection of Industrial and MarineBacteria (23 St Machar Drive, Aberdeen, Scotland, UK, AB2 1RY) underaccession number NCIMB 40795. This deposit was made under the provisionsof the Budapest Treaty on Apr. 19, 1996.

This microorganism is particularly preferred because it produces inaddition to an amidase enzyme suitable for use in the process of theinvention a nitrile hydratase enzyme. This acts non-enantioselectively.Thus using this single microorganism Rhodococcus NCIMB 40795enantioselective conversion of α-amino nitrile to α-amino acid viaα-amino amide can be conducted.

This microorganism is in itself a new strain of microorganism. Thereforeaccording to a second aspect of the invention we provide a microorganismwhich is Rhodococcus NCIMB 40795 or a mutant thereof having the abilityto produce an amidase. This new microorganism is useful for theconversion of nitrites and amides to α-amino acids. According to a thirdaspect of the invention we also provide a process of converting anα-amino amide to an α-amino acid comprising conducting a conversionreaction catalyzed by an amidase enzyme, in which process the α-aminoamide starting material comprises amide enantiomers (A) and (B) and inthe conversion reaction enantiomer (A) is converted preferentially overenantiomer (B), characterized in that the amidase enzyme is produced byRhodococcus NCIMB 40795. This process preferably has any or all of thefeatures discussed above in connection with the process of the firstaspect of the invention.

The Rhodococcus strain of the invention is believed to be an atypicalstrain of this genus. It is Gram-Positive, gives no spores and isnon-motile. It grows well at 30° C. and less well at 37° C. and 41° C.but not at 45° C. It shows catalyze activity but not oxidase activityand is not fermentative in glucose OF.

The cell wall diamino acid is meso DAP. Mycolic acids are present. Thefatty acid profile includes the following:

tetradecanoic acid, pentadecanoic acid, hexadecenoic acid, hexadecanoicacid, heptadecenoic acid, heptadecanoic acid, octadecenoic acid but verylittle tuberculostearic or other 10 methyl branched acids (less than 1%of each).

The invention also provides new enantioselective amidase enzymes. Anamidase enzyme of the fourth aspect of the invention is obtainable byculturing Rhodococcus NCIMB 40795 or a mutant thereof capable ofproducing an amidase.

We have also found that a second specific microorganism strain isparticularly useful in carrying out the process of the invention. Thisis a strain of the species Rhodococcus wratslaviensis deposited at theNational Collection of Industrial and Marine Bacteria under accessionnumber NCIMB 13082, named as Tsukamurella wratslaviensis. Thismicroorganism produces an amidase which can carry out the process of theinvention.

The invention also provides processes in which α-methyl amides andα-aminomethyl amides are converted to their corresponding acids. Thusaccording to a fifth aspect of the invention we provide a process ofconverting an α-methyl amide or an α-aminomethyl amide to an α-methylacid or an α-aminomethyl acid, comprising conducting a conversionreaction catalyzed by an amidase enzyme, in which the α-methyl amide orα-aminomethyl amide starting material comprises amide enantiomers (A)and (B) and in the conversion reaction enantiomer (A) is convertedpreferentially over enantiomer (B),

characterized in that the amidase enzyme is capable of convertingenantiomer (A) such that it gives an enantiomeric excess of at least 90%independently of the conversion time.

In this process of the fifth aspect of the invention the α-methyl amideis suitably of the formula III as follows:

in which R may be any of the groups discussed above for R in formula I.

The α-aminomethyl amide suitably has the formula IV as follows:

in which R has any of the values suggested for R in formula I above.

The conversion reactions of the α-methyl amide and α-aminomethyl amideproduce α-methyl acids and α-aminomethyl acids respectively, of theformulae V and VI, as follows:

Any of the features of the process of the first aspect of the inventionmay be applied to the process of the fifth aspect of the invention.

We find that the α-methyl amides and α-aminomethyl amides tend to beconverted, especially by the amidase from the microorganism RhodococcusNCIMB 40795, at a slower rate than the α-amino acids.

Products of any of the processes of the invention are highlyenantiomerically pure. They are particularly useful as intermediates inthe preparation of pharmaceuticals and agrochemicals, for instanceantibiotics.

The following are some examples of the invention.

EXAMPLE 1 Culture of Microorganism

The original isolate Rhodococcus NCIMB 40795 was cultured using 500 mlshake flasks, with orbital shaking (200 rpm) at 30° C.

The culture medium was prepared as shown below, with all quantities ing/l unless otherwise stated:

Dipotassium hydrogen phosphate 7.0 Potassium dihydrogen phosphate 3.0Sodium acetate 5.0 Propionitrile 714 μl Magnesium sulphate* 1.0 Calciumchloride* 0.2 [*autoclaved separately] Vitamins (at 1 ml/l g/l Thiaminehydrochloride 0.1 Calcium pantothenate 0.1 Pyridoxine hydrochloride 0.1Biotin 0.01 Inositol 10.0 Trace metals (at 5 ml/l) g/l MgO 10.75 CaCO₃2.0 FeSO₄.7H₂O 4.5 ZnSO₄.7H₂O 1.44 MnSO₄.4H₂O 1.12 CuSO₄.5H₂O 0.25CaSO₄.7H₂O 0.28 H₃BO₃ 0.06 HCl (6 N) 51.3 cm³

The cells were harvested in late exponential growth by centrifugation at10,000 rpm for 20 minutes. The cell pellet was washed with saline (250ml) and centrifuged again under the same conditions.

The cell pellet was then frozen until needed for use in the conversionreaction.

EXAMPLE 2 Conversion of α-Amino Nitrile

Phenylglycinonitrile (16.8 mg, 0.1 mmol) was dissolved in phosphatebuffer (10 ml, pH 7.0, 50 mM) to give a 10 mM concentration. Thesolution was incubated with whole cells produced as described in Example1(at 4.2 g/L [dry cells] at 20° C. and samples taken as outlined below:

A 50 μl sample was removed and diluted with 450 μl water. The cells wereremoved by microcentrifugation (13,000 rpm, 30 seconds). The supernatantwas decanted off, and HCl (10 μl, 6N) was added The mixture wasextracted with dichloromethane to removed any aldehyde formed by thedegradation of the nitrile, which would interfere with the HPLC. Theaqueous phase was then analysed by HPLC.

All HPLC was run using a LDC/Milton Ray ConstaMetric 3 with a UVdetector.

Column Phenomenex RP select B (25 cm × 0.46 cm) Flow rate 1 ml min⁻¹Wavelength 254 nm Eluent 95% Tris/HCl buffer (10 mM, pH 3): 5% methanolRetention Acid 6.7 minutes, Amide 8.0 minutes, Nitrile 13.6 minutes

After 10% conversion, a sample (200 μl) was taken and extracted withdichloromethane to remove any unreacted nitrile. This was refluxed inHCl (6N) until HPLC showed that all the amide had been converted toacid. The acid was then subjected to chiral HPLC as outlined below:

Column Sumichiral OA 5000 (15 cm × 0.46 cm) Flow rate 1 ml min⁻¹Wavelength 254 nm Eluent 90% CuSO₄ (3 mM): 10% methanol RetentionEnantiomer 1:- 18 minutes, Enantiomer 2:- 30 minutes

The amide produced was found to be racemic, i.e. the nitrile hydrataseis not enantioselective.

In this case the reaction was inhibited by cyanide produced by thedegradation of the nitrile. However it was possible to achieve 100%conversion of the nitrile by adding more cells.

EXAMPLE 3 Conversion of α-Amino Amide

4 Chloro phenyl α-amino acetamide (11 mg, 0.05 mmol) was dissolved inphosphate buffer (5 ml, pH 7.0, 50 mM) to give a 10 mM concentration.The solution was incubated at 30° C. and the whole cells produced asdescribed in Example 1 were added (at 4.2 g(dry cells) /1). Samples weretaken at various time intervals as outlined below:

Sample at time=0 mins:

1. A 50 μl sample was removed and diluted with 450 μl water. The cellswere removed by microcentrifugation (13,000 rpm, 30 seconds) and thesample analysed by HPLC.

Column Phenomenex RP select B (25 cm × 0.46 cm Flow rate 1 ml minWavelength 254 nm Eluent 80% Tris/HCl buffer (10 mM, pH 3): 20% methanolRetention Acid 6.7 minutes, Amide 8.0 minutes

2. A 200 μl sample was removed and centrifuged as above. This wassubjected to chiral HPLC as outlined below:

Column Sumichiral OA 5000 (15 cm × 0.46 cm) Flow rate 1 ml min⁻¹Wavelength 254 nm Eluent 75% CuSO₄ (3 mM): 25% methanol RetentionEnantiomer 1: − 25 minutes, Enantiomer 2: − 40 minutes.

Samples were analysed subsequently at various times by method 1 until itwas observed that 50% conversion of the amide had been achieved. At thispoint sampling methods 1 and 2 were again used.

It was observed that the concentration of amide decreased from 10 mM toaround 5.0 mM over a period of around 140 minutes. Over the same periodthe concentration of 4-chlorophenyl α-amino acetic acid increased from 0to around 5.0 mM. After around 145 minutes it could be seen that thenatural termination of the reaction had occurred as the rate was thensubstantially zero.

The same procedure was adopted with other substituted and unsubstitutedphenyl α-amino acetamides and with cyclic and acyclic aliphatic amides.Results are shown in Table 1 below.

General Formula I

R % Conversion % e.e. para-H-phenyl 50 >98 para-CH₃-phenyl 50 >98meta-CH₃-phenyl 50 >98 ortho-CH₃-phenyl 50 >98 para-CF₃-phenyl 50 >98para-Et-phenyl 50 >98 para-(CH₃)₃C-phenyl 50 >98 para-Cl-phenyl 5o >98para-CH₃(CH₂)₃O-phenyl 50 NOT DETERMINED para-OH-phenyl 50 >98 C₄H₉50 >98 C₉H₁₅ 50 >98

50 >98

50 >98 (CH₃)₂CHCH₂ 50 >98 para-CH₃O-phenyl 50 >98

50 >98

EXAMPLE 4

α-phenyl α-amino acetamide (75 mg, 0.5 mmol) was dissolved in phosphatebuffer (5 ml, pH 7.0, 50 mM) to give a 10 mM concentration. The solutionwas incubated at 30° C. and the whole cells produced as described inExample 1 were added (at 4.2 g (dry cells)/L) Samples were taken atvarious time intervals as outlined below.

Sample at time=0 mins

1. A 45 μl sample was removed and diluted with 450 μl water. The cellswere removed by microcentrifugation (13,000 rpm, 30 seconds) and thesample analysed by HLPC.

Column: Phenomenex RP Select B (25 cm × 046 cm) Flow Rate: 1 ml min⁻¹Wavelength: 254 nm Eluent: 95% Tris/HCl buffer (10 mM, pH 3): 5%methanol Retention: Acid 4.7 minutes, Amide 5.8 minutes

2. A 200 μl sample was removed, centrifuged as above. This was subjectedto chiral HPLC as outlined below.

Column: Sumiiral OA 5000 (15 cm × 0.4 cm) Flow Rate: 1 ml min⁻¹Wavelength: 254 nm Eluent: 90% CuSO4 (2 mM): 10% methanol Retention: LEnantiomer of α-phenyl α-amino acetic acid - 18 minutes D Enantiomer ofα-phenyl α-amino acetic acid - 30 minutes

Samples were analysed subsequently at various times by method 1 until itwas observed that 50% conversion of the amide had been achieved. At thispoint sampling methods 1 and 2 were used.

It was observed that the concentration of amide decreased from 10 mM toaround 5.0 mM over a period of 24 hours. Over the same period theconcentration of α-phenyl α-amino acetic acid increased from 0 to around5 mM. After 48 hours the concentrations of both the amide and theα-phenyl α-amino acetic acid were still at around 5 mM. Therefore, ataround 24 hours incubation time it could be seen that the naturaltermination of the reaction had occurred.

EXAMPLE 5

Whole cells produced as described in Example 1 were added (at 4.2 g (drycells)/L) to 32 ml of 50 mM, pH 7 sodium phosphate buffer at 35° C.α-phenyl α-amino acetamide solution (8 ml of 50 mM) was added to thecell suspension to give a concentration of 10 mm α-phenyl α-aminoacetamide. Samples were taken at various time intervals as outlinedbelow:

Sample time=0 minutes

1. A 0.5 ml sample was removed and the cells removed bymicrocentrifugation (13,000 rpm, 30 seconds). The supernatant wasdiluted by a factor of 5 and analysed by HPLC using the conditionsdescribed in Example 4.

2. A 200 μl sample was removed, centrifuged as above. The supernatantwas subjected to chiral HPLC as outlined in Example 3. Samples wereanalysed subsequently at 20 minute intervals by method 1. After 4 hoursit was observed that 30% conversion of the amide had been achieved. Atthis point sampling methods 1 and 2 were used.

It was observed after 4 hours that the concentration of amide haddecreased from 10 mM to around 7 mM and that the concentration ofα-phenyl α-amino acetic acid had increased from o to 3 mM. When thesupernatant was analysed by method 2 it was seen that upon 30%conversion of the amide substantially a >98% enantiomeric excess of theL enantiomer of α-phenyl α-amino acetic acid was achieved.

A further 6 ml of 50 mM α-phenyl α-amino acetamide was then added to thecell suspension so that the cell suspension then contained whole cellsat 3.57 g(dry cells)/L, 2.55 mM of the enantiomer of α-phenyl α-aminoacetic acid and 13.45 mM of amide.

It was observed that the concentration of amide had decreased from 13.45mM to 10 mM and that the concentration of α-phenyl α-amino acetic acidhad increased from 2.55 to 6 mM. When the supernatant was analysed bymethod 2 it was seen that substantially a >98% enantiomeric excess ofthe L enantiomer of α-phenyl α-amino acetic acid was achieved.

EXAMPLE 6

Whole cells produced as described in Example 1 were combined with 10 mMα-phenyl α-amino acetamide under conditions as described in Example 5.Samples were taken at various time intervals and analysed as describedin Example 5.

It was observed that after 90 minutes the concentration of α-phenylα-amino acetic acid had increased from 0 to 3.24 mM. At this pointsufficient quantity of α-phenyl α-amino acetamide was added to raise itsconcentration in the reaction mixture by 10 mM. It was observed thatafter a further 90 minutes, the concentration of α-phenyl α-amino aceticacid had increased to 5.86 mM. At this point sufficient quantity ofα-phenyl α-amino acetamide was added to raise its concentration in thereaction mixture by 10 mM. Upon analysis by method 1. It was observedthat after a further 90 minutes, the concentration of α-phenyl α-aminoacetic acid had increased to 7.98 mM. The cells were then separated fromthe reaction mixture by centrifugation at 13000 g for 10 minutes. Whenthe supernatant was analysed by method 2 it was seen that substantiallya >98% enantiomeric excess of the L enantiomer of α-phenyl α-aminoacetic acid was achieved.

EXAMPLE 7

To the 17.26 g of final supernatant produced in Example 6, was added20.74 g of water to reduce L-α-phenyl α-amino acetic acid concentrationto 3.70 mM. To this solution 0.65 g of whole cells produced as describedin Example 1 was added.

It was observed that after 45 minutes the concentration of α-phenylα-amino acetic acid had risen to 3.75 mM. At this point a sufficientquantity of α-phenyl α-amino acetamide was added to the reaction mixtureto raise its concentration in the reaction mixture by 12 mM. It wasobserved that after a further 120 minutes the concentration of α-phenylα-amino acetic acid had increased to 7.98 mM. The procedure of addingfurther α-phenyl α-amino acetamide followed by 2 hours incubation asdescribed above was repeated twice again. Upon analysis by method 1, itwas observed that the concentration of α-phenyl α-amino acetic acid hadincreased to 14.81 mM. The cells were then separated from the reactionmixture and analysed by method 2 as described in Example 6. It was seenthat substantially a >98% enantiomeric excess of the L enantiomer ofα-phenyl α-amino acetic acid was achieved.

EXAMPLE 8

Whole cells produced as described in Example 1 (0.52 g) were added tothe final supernatant produced in Example 7.

It was observed that after 40 minutes of incubation at 35° C. theconcentration of α-phenyl α-amino acetic acid had increased to 17.46 mM.

On three separate occasions sufficient quantity of α-phenyl α-aminoacetamide was added to the reaction mixture to raise its concentrationby 12 mM. After each addition the reaction mixture was allowed toincubate for 120 minutes. Upon analysis by method 1 of the reactionmixture and after the completion of the procedure described above, itwas observed that the concentration of α-phenyl α-amino acetic acid hadincreased to 26.64 mM. The cells were then separated from the reactionmixture and analysed by method 2 as described in Example 6. It was seenthat substantially a >98% enantiomeric excess of the L enantiomer ofα-phenyl α-amino acetic acid was achieved.

EXAMPLE 9

Whole cells produced as described in Example 1 (0.48 g) were suspendedin the final supernatant produced in Example 8.

On two separate occasions sufficient quantity of α-phenyl α-aminoacetamide was added to the reaction mixture to raise its concentrationby 10 mM. After each addition the reaction mixture was allowed toincubate at 35° C. for 120 minutes. Upon analysis by method 1 of thereaction mixture and after the completion of the procedure above, it wasobserved that the concentration of α-phenyl α-amino acetic acid hadincreased to 30.38 mM. The cells were then separated from the reactionmixture and analysed by method 2 as described in Example 6. It was foundthat substantially a >98% enantiomeric excess of the L enantiomer ofα-phenyl α-amino acetic acid was achieved.

EXAMPLE 10

Whole cells produced as described in Example 1 (1.07 g) were suspendedin 36.9 ml of the L enantiomer of α-phenyl α-amino acetic acid(purchased from Aldrich).

Sufficient quantity of α-phenyl α-amino acetamide was then added to givean α-phenyl α-amino acetamide concentration of 50 mM and dilute the Lenantiomer of α-phenyl α-amino acetic acid to 40 mM. The reactionmixture was then allowed to incubate at 30° C. for 300 minutes. After300 minutes it was observed that an approximately 30% conversion of theamide had been achieved. At this point sampling methods 1 and 2 wereused.

It was observed after 300 minutes that the concentration of α-phenylα-amino acetic acid had increased from 40 mM to 52.6 mM. When thesupernatant was analysed by method 2, it was seen that upon anapproximately 30% conversion of the amide, substantially a >98%enantiomeric excess of the L enantiomer of α-phenyl α-amino acetic acidwas observed.

After a further 960 minutes incubation at 35° C., it was observed that a40% conversion of the amide had been achieved. At this point samplingmethods 1 and 2 were used.

It was observed after 960 minutes that the concentration of α-phenylα-amino acetic acid had increased to 58.3 mM. When the supernatant wasanalysed by method 2, it was seen that upon an approximately 40%conversion of the amide, substantially a >98% enantiomeric excess of theL enantiomer of α-phenyl α-amino acetic acid was observed.

These results show the excellent enantiomeric excess which can beachieved using the invention. In particular it will be seen that 50%conversion of the starting material, ie substantially 100% of enantiomer(A), is achieved. This result is also achieved over a wide range ofα-amino amides. Such results can be achieved also in the presence oflarge excesses of the unconverted enantiomer B (in this case the Denantiomer) and in the presence of high concentrations of the productacid.

What is claimed is:
 1. A process of converting an α-amino amide to anα-amino acid comprising conducting a conversion reaction catalyzed by anamidase enzyme, in which the α-amino amide starting material comprisesamide enantiomers (A) and (B) and in the conversion reaction enantiomer(A) is converted preferentially over enantiomer (B), characterized inthat the amidase enzyme is produced by Rhodococcus NCIMB 40795 orRhodococcus wratslaviensis NCIMB 13082, and in which the amidase enzymeis capable of converting enantiomer (A) such that it gives anenantiomeric excess of at least 90% independently of the conversiontime.
 2. A process according to claim 1 in which the amidase enzyme iscapable of converting enantiomer (A) such that it gives an enantiomericexcess of at least 98% independently of the conversion time.
 3. Aprocess according to claim 1 in which the α-amino amide startingmaterial is a racemic mixture of enantiomers (A) and (B).
 4. A processaccording to claim 1 in which the enantiomer (B) is present in excessover enantiomer (A) for at least 30 minutes during the conversionreaction.
 5. A process according to claim 4 in which the amount ofenantiomer (B) is at least 150% of the amount of enantiomer (A) for atleast 30 minutes during the conversion reaction.
 6. A process accordingto claim 1 in which the α-amino amide has the formula I:

in which R is alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, alkaryl;aralkyl, R¹NHCOR¹, R¹CONHR¹, SO₂R¹ or SO₂NHR¹ in which R¹ is alkyl,cycloalkyl, alkenyl, cycloalkenyl, aryl, alkaryl or aralkyl, orsubstituted versions of any of these, preferably C₄ to C₉ linear orbranched alkyl or alkenyl, cyclic alkyl or alkenyl, phenyl, orsubstituted phenyl in which the substituent is selected from para-CH₃,meta-CH₃, ortho-CH₃, para-CF₃, para-Et, para-(CH₃)₃C, para-Cl,para-CH₃(CH₂)₃O and para-OH.
 7. A process according to claim 1 in whichthe α-amino acid is an unnatural amino acid.
 8. A process according toclaim 1 in which the enantiomer (A) is the L-enantiomer and is convertedinto the L α-amino acid.
 9. A process according to claim 1 in which anenantiomeric excess of at least 98% is given independently of theconversion time.
 10. A process according to claim 1 which is a fed batchprocess.
 11. A process according to claim 1 in which the conversionreaction has a duration of at least 12 hours.
 12. A process according toclaim 1 in which the α-amino acid corresponding to enantiomer (A) ispresent in the reaction mixture in excess over enantiomer (A) for atleast 30 minutes during the conversion reaction.
 13. A process accordingto claim 4 in which the ratio of α-amino acid corresponding toenantiomer (A) to enantiomer (A) itself is at least 3:2.
 14. A processof converting an α-amino amide to an α-amino acid comprising conductinga conversion reaction in which the α-amino amide starting materialcomprises amide enantiomers (A) and (B) and in the conversion reactionenantiomer (A) is converted preferentially over enantiomer (B),characterized in that the conversion is brought about by contacting thestarting material with cell material from Rhodococcus NCIMB 40795 orRhodococcus wratslaviensis NCIMB
 13082. 15. A process of converting anα-methyl amide or an α-aminomethyl amide to an α-methyl acid or anα-aminomethyl acid, comprising conducting a conversion reactioncatalyzed by an amidase enzyme, in which the α-methylamide orα-aminomethyl amide starting material comprises amide enantiomers (A)and (B) and in the conversion reaction enantiomer (A) is convertedpreferentially over enantiomer (B), characterized in that the amidaseenzyme is produced by Rhodococcus NCIMB 40795 or Rhodococcuswratslaviensis NCIMB
 13082. 16. A process of converting an α-methylamide or an α-aminomethyl amide to an α-methyl acid or an α-aminomethylacid, comprising conducting a conversion reaction catalyzed by anamidase enzyme, in which the α-methylamide or α-aminomethyl amidestarting material comprises amide enantiomers (A) and (B) and in theconversion reaction enantiomer (A) is converted preferentially overenantiomer characterized in that the amidase enzyme is capable ofconverting enantiomer (A) such that it gives an enantiomeric excess ofat least 90% independently of the conversion time characterized in thatthe conversion is brought about by contacting the starting material withcell material from Rhodococcus NCIMB 40795 or Rhodococcus wratslaviensisNCIMB 13082.